Introduction
Toxicity is a complex event in vivo and exhibits a wide spectrum of effects from cell death to metabolic aberrations, which result in functional changes in cells rather than death.
A. Assays based on cell viability and cell survival
Traditionally, Cytotoxicity assays have been based on indices of cell viability and survival. Viability is an instantaneous parameter thought to be predicted of survival.
Cytotoxicity assays can be divided into three groups:
1. Those that infer viability by determination of a change in membrane permeability or metabolism.
2. Those that determine viability trough absolute long-term survival, as quantified by the capacity to regenerate (Clonogenicity.)
3. Those that determine survival in an altered state by expressing a generic mutation or malignant transformation.
Viability assays
Viability assays include:
Dye exclusion methods.
Dye uptake methods.
Fluorescent methods.
Dye release methods.
Dye exclusion methods: (more…)
Chromosome walking is a method used to clone in an orderly fashion the DNA segments along the chromosome starting at any point for which we have a probe. To build up a series of overlapping cloned DNA fragments, it begins with one clone from a library, and then identifies a second clone whose insert overlaps with the insert in the first clone. This was the first method, called chromosome walking, devised for assembly of clone contigs. But there is a limitation to the speed of chromosome walking and that is because of the small size of the fragments that are to be cloned, another limitation is the difficulty of walking through the repeated sequence that are scattered through the gene. A more straightforward approach thus is to use the insert DNA from the starting clone as a hybridization probe to screen all the other clones in the library. Positive hybridization signals that are given by clones, whose inserts overlap with the probe, are used as new probes to continue the walk. There are about 96 clones that a library consists of and each clone contains a different insert. The insert from one of the selected clones is then used as a hybridization probe with all other clones in the library.